Biological properties and the nutrition value of an Isochrysis strain as a live food for geo-duck larvae Le Thi Phuong Hoa 1 , Nguyen Thi Hoai Ha 2 , Pham Thi Bich Dao 2 , Lưu Thị Thùy Giang 2 , Luong Thanh Hao 3 1 Faculty of Biology, Hanoi National University of Education 2 Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi 3 Faculty of Biology, College of Natural Sciences Abstract The addition of Isochrysis galbana H5 ensures a balanced supply of the omega3 and omega6 fatty acids for the survival and healthy of geoduck larvae. This study has shown that the critical factor in this diet for geoduck larvae is the content of polyunsaturated fatty acids (PUFAs), accounted for more than 40 % of total fatty acids, and docosahexaenoic acid (DHA- 5.53 mg per mg fresh weight), which play an important role in the membrane lipids. Medium f/2 was selected as best medium for microalgae culture. Isochrysis galbana H5 is recommended as a live food for growing larvae and juvenile of fish, bivalves and crustaceans in aquaculture. Keyword: fatty acid, marine microalgae, geoduck 1. Introduction Marine microalgae provide the food base which supports the entire animal population of the open sea. They contain a wide range of fatty acids especially polyunsaturated fatty acids (PUFAs), which cannot be produced in sufficent quantities for metabolic functioning in most marine animals [3,5,12]. Many microalgae are believed to improve the nutritional conditions of the larvae as well as their growth and survival [7,12]. They are widely used in aquatic cultivation as food for bivalves, crustaceans and fish especially in their larval stage [12]. The prymnesiophyte Isochrysis is the one of the most commonly used in mariculture, which is rich in essential fatty acids, omega6-linoleic acid (C18:2n-6) and omega3-linolenic acid (C18:3n-3), and the highly polyunsaturated omega3 fatty acids, octadecatetraenoic acid (C18:4n-3), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) [2,10,12]. Geo-duck is a bivalve species, which inhabits commonly in Quang Ninh and is served as a specialty with high nutrition [6]. In this study, Isochrysis strains isolated, in a way to find suitable food for sustainable development and conservation of geo-ducks in their larval stage. 2. Materials and Methods Isochrysis strains were isolated based on their morphological properties using micropipettes [9] and jellylike desert dish according to Shirai et al. [8]. They were grown at room temperature and illuminated under neon light (Philips daylight tubes) on 10: 14 h light: dark cycles. Media f/2, ASW and ESM [4] were used to select culture medium for the growth and development. Cells were cultured in 250 ml conical flask and harvested every two days. Cell density was determined with a Neubauer haemocytometer in three replicates. Total DNA was extracted and 18S rDNA-coding region were amplified and sequenced according to An et al. [1]. The 18S rDNA sequences were analyzed using BLAST tool. Biomass for fatty acid analysis was obtained from 1-4 liter aerated culture suspensions in plastic containers. Cells were harvested at the log phase to get healthy cells. Fatty acid composition was determined according to Krienitz et al. [5] by gas chromatography (Finnigan Trace GC) using an ultra-column BPX70. Fatty acids were identified by comparing retention times with those of a calibration standard solution. 3. Results and discussion 3.1. Isolation, and classification of Isochrysis strains Isochrysis strains were isolated based on their morphological properties. One of them is signified as H5 (Fig. 1). Figure 1. Microscopic morphology of Isochrysis H5 Figure 2. Agarose gel electrophoresis of 18S rDNA SSU rDNA sequence comparison has proved to be a powerful alternative to morphology for inferring phylogenetic relationships at all taxonomic levels. The result on analysis and alignment of 18S rDNA sequences confirms Isochrysis H5 identification as Isochrysis galbana. Figure 2 shows the18S rDNA amplification products The phylogenetic tree constructed from neighbour – joining analysis of the sequences of 18SrDNA depiciting relationships of species (Figure 3). 3.2. Selection of culture medium Optimization of culture conditions for the selected strains is essential for algal mass culture. Furthermore, growth characteristics have been shown to have a significant impact on the lipid and fatty acid profiles [1,7]. In this study, medium f/2, ASW and ESM were used. The cells were collected every two days and counted (Table 1). H5 Isochrysis galbana Gephyrocapsa oceanica Emiliania huxleyi Coccoid haptophyte 88 100 100 Phaeocystis antarctica 96 Prymnesium nemamethecum Prymnesium patelliferum Chrysochromulinda polylepis 100 Chrysochromulina hirta 100 Coccolithus pelagicus Coccolithus braarudii Cruciplaccolithus neohelis 100 Pleurochrysis carterae Pleurochrysis carterae 100 100 87 73 0.005 100 Figure 3. The phylogenetic tree constructed from neighbour – joining analysis of the sequences of 18SrDNA Table 1. Cell density of Isochrysis galbana grown on different media Time (days) Cell density (x 10 6 /ml) F/2 ESM ASW 0 0.7 0.7 0.7 2 0.65 1.05 0.8 4 1.75 1.45 1.1 6 3.75 1.75 1.35 8 4.05 1.8 1.9 10 7.15 2.35 2.35 12 5.65 2.05 2.7 14 5 1.85 2.1 16 4.6 1.55 1.75 18 2.1 0.62 0.76 Isochrysis galbana H5 grew the better in f/2 than in the ESM media and ASW media. The growth of Isochrysis galbana H5 reached the highest peak after 10 days culture (7.15 x 10 6 cell/ml). It is suggested that f/2 is the most suitable for culturing Isochrysis galbana H5 and can be applied for biomass production in aquaculture. 3.3. Fatty acid composition Lipid has its own specificity in each species of living organisms. Fatty acids are mostly located in the cell membrane, which is much enough to apply to taxonomic characterization [3,12]. Fatty acid components of Isochrysis galbana H5 were analysed (Table 2). Table 2. Composition of fatty acids in Isochrysis galbana H5 Fatty acid Chemical name Quantities (mg/1mg fresh weight) C14:0 Myristic acid 4.16 C16:0 Palmitic Acid 14.69 C16:1 Palmitoleic Acid 2.09 C18:0 Stearic Acid 1.22 C18:1n9 Oleic Acid 5.42 C:18:2n6 Linoleic Acid 2.71 C:18:3n3 Anpha-Linoleic Acid 3.52 C18:4n3 Octadecatetraenoic 18.9 C:20:5n3 Eicosapentaenoic Acid (EPA) 0.24 C22:0 Benhenic acid 15.89 C:22:6n3 Docosahexaenoic Acid (DHA) 5.53 The total fatty acids in Isochrysis galbana H5 are 74.37 g/mg fresh weight. Isochrysis galbana H5 has a great variety of fatty acids ranging from 14C to 22C, among C16:0, C18:4n-3 and C22:0 are the most abundant fatty acids. The compossition is different from previous report [10] but major variations in the fatty acid compositions of Isochrysis galbana have been reported [12]. More than 40 % of total fatty acids (30.9 g/mgfresh weight) in Isochrysis galbana H5 is polyunsaturated fatty acids, suggesting the high quality of Isochrysis galbana H5 and the potential to be applied in aquatic food webs such as for feeding geo-ducks. In addition, DHA had remarkably high content (5.53 g) while EPA was at low abudance, smilar to Isochrysis sp. Clone T.ISO in previous report [12]. DHA and EPA play an important role in the membrane lipids. 3.4. Application of Isochrysis galbana H5 for feeding geo-duck at Vandon, Quangninh Isochrysis galbana H5 was cultured in F/2 medium, grown at room temperature and illuminated under neon light (Philips daylight tubes) 4000 - 5000 Lux as the following scheme with illustrated pictures Microalgae volume was used depending on geo-duck larvae density.Geoduck larvae with 2.5x10 5 -16.6x10 5 individuals per m 3 were fed in 120 – 180 liters microalage with cell density 7.7 - 8.2 10 6 cells/ml per day . Conclusions The Isochrysis strain were successfully isolated from Vandon, Quangninh and identified as Isochrysis galbana. The suitable medium for strain was f/2. The Isochrysis galbana strain showed a huge range of fatty acids among, contained remarkable amount of PUFA and considerate level of EPA and DHA which play an essential role in cell membrane physiology and hormone metabolism, suggesting high value as food for amimals, specially for feeding geo-duck larvae. Stock culture Cell density 1.25- 2.1 10 5 cells/ml 2-6 litre flask After 5-7 days, cell density 4.2 10 6 cells/ml 30–60 litre plastic bag Cell density 5.7- 7.15 10 6 cells/ml after 4-6 days 160 litre tank After 4-6 days, cell density 7.7 - 8.2 10 6 cells/ml 10 litre container After 2-3 days, cell density 5.3-6.4 10 6 cells/ml References 1. S.S. An, T. Friedl, E. Hegewald, “Phynogenetic relationships of Scenedesmus and Scenedesmus–like coccoid green algae as inferred from ITS–2 rDNA sequence comparisons”, Plant Biology, Vol. 1, No 4, (1999), pp. 418 – 428. 1. 2. Ide Ni Fhaolain & Stephen Fitzpatrick, “Marine microalgae as a source of omega3 fatty acids” Galway-Mayo Institute of Technology, The Irish Scientist No7 (1999) 3. IA. Guschina, J. L. Harwood. Lipids and lipid metabolism in eukaryotic algae. Progress in Lipid Research, Vol. 45, (2006), pp. 160–186. 4. F.Kasai, M.Kawachi, et al., Nies- Collection List of Straine, 8th Edition, Japan (2009) 5. L. Krienitz, M. Wirth. 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Freshwater of North America: Ecology and classification. 2003. Elsevier Inc. 12. J. K. Volkman, S. W. Jeffrey, P. D. Nichols, G. I. Rogers and C. D. Garland. Fatty acid and lipid composition of 10 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology, Vol. 128, (1989), pp. 219-240. . suitable medium for strain was f/2. The Isochrysis galbana strain showed a huge range of fatty acids among, contained remarkable amount of PUFA and considerate level of EPA and DHA which play an. Natural Sciences Abstract The addition of Isochrysis galbana H5 ensures a balanced supply of the omega3 and omega6 fatty acids for the survival and healthy of geoduck larvae. This study has. shirai, Katsumi Matumaru, Akio Ohotake, Yoshichika Takamura, Tokujiro Adia and Masayasu Nakano (1989), “Development of a Solid medium for Growth and Isolation of Axenic Microcystis Strains (Cyanobacteria)